Short-distance vesicle transport via phase separation.

In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca2+ and via its C2A domain-mediated Ca2+ sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.

Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and Plasticity.

Synapses are semi-membraneless, protein-dense, sub-micron chemical reaction compartments responsible for signal processing in each and every neuron. Proper formation and dynamic responses to stimulations of synapses, both during development and in adult, are fundamental to functions of mammalian brains, although the molecular basis governing formation and modulation of compartmentalized synaptic assemblies is unclear. Here, we used a biochemical reconstitution approach to show that, both in solution and on supported membrane bilayers, multivalent interaction networks formed by major excitatory postsynaptic density (PSD) scaffold proteins led to formation of PSD-like assemblies via phase separation. The reconstituted PSD-like assemblies can cluster receptors, selectively concentrate enzymes, promote actin bundle formation, and expel inhibitory postsynaptic proteins. Additionally, the condensed phase PSD assemblies have features that are distinct from those in homogeneous solutions and fit for synaptic functions. Thus, we have built a molecular platform for understanding how neuronal synapses are formed and dynamically regulated.

Phosphorylation-dependent membraneless organelle fusion and fission illustrated by postsynaptic density assemblies.

Membraneless organelles formed by phase separation of proteins and nucleic acids play diverse cellular functions. Whether and, if yes, how membraneless organelles in ways analogous to membrane-based organelles also undergo regulated fusion and fission is unknown. Here, using a partially reconstituted mammalian postsynaptic density (PSD) condensate as a paradigm, we show that membraneless organelles can undergo phosphorylation-dependent fusion and fission. Without phosphorylation of the SAPAP guanylate kinase domain-binding repeats, the upper and lower layers of PSD protein mixtures form two immiscible sub-compartments in a phase-in-phase organization. Phosphorylation of SAPAP leads to fusion of the two sub-compartments into one condensate accompanied with an increased Stargazin density in the condensate. Dephosphorylation of SAPAP can reverse this event. Preventing SAPAP phosphorylation in vivo leads to increased separation of proteins from the lower and upper layers of PSD sub-compartments. Thus, analogous to membrane-based organelles, membraneless organelles can also undergo regulated fusion and fission.

Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity.

Postsynaptic densities (PSDs) are membrane semi-enclosed, submicron protein-enriched cellular compartments beneath postsynaptic membranes, which constantly exchange their components with bulk aqueous cytoplasm in synaptic spines. Formation and activity-dependent modulation of PSDs is considered as one of the most basic molecular events governing synaptic plasticity in the nervous system. In this study, we discover that SynGAP, one of the most abundant PSD proteins and a Ras/Rap GTPase activator, forms a homo-trimer and binds to multiple copies of PSD-95. Binding of SynGAP to PSD-95 induces phase separation of the complex, forming highly concentrated liquid-like droplets reminiscent of the PSD. The multivalent nature of the SynGAP/PSD-95 complex is critical for the phase separation to occur and for proper activity-dependent SynGAP dispersions from the PSD. In addition to revealing a dynamic anchoring mechanism of SynGAP at the PSD, our results also suggest a model for phase-transition-mediated formation of PSD.

Transitions in the framework of condensate biology.

As phase separation is found in an increasing variety of biological contexts, additional challenges have arisen in understanding the underlying principles of condensate formation and function. We spoke with researchers across disciplines about their views on the ever-changing landscape of biomolecular condensates.

Structural and mechanistic insights into fungal ß-1,3-glucan synthase FKS1.

The membrane-integrated synthase FKS is involved in the biosynthesis of ß-1,3-glucan, the core component of the fungal cell wall1,2. FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp3,4. Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane-cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1-membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5-6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal ß-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS.

Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates.

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy.

GIT/PIX Condensates Are Modular and Ideal for Distinct Compartmentalized Cell Signaling.

Enzymes or enzyme complexes can be concentrated in different cellular loci to modulate distinct functional processes in response to specific signals. How cells condense and compartmentalize enzyme complexes for spatiotemporally distinct cellular events is not well understood. Here we discover that specific and tight association of GIT1 and ß-Pix, a pair of GTPase regulatory enzymes, leads to phase separation of the complex without additional scaffolding molecules. GIT1/ß-Pix condensates are modular in nature and can be positioned at distinct cellular compartments, such as neuronal synapses, focal adhesions, and cell-cell junctions, by upstream adaptors. Guided by the structure of the GIT/PIX complex, we specifically probed the role of phase separation of the enzyme complex in cell migration and synapse formation. Our study suggests that formation of modular enzyme complex condensates via phase separation can dynamically concentrate limited quantities of enzymes to distinct cellular compartments for specific and optimal signaling.

Critical roles of phosphoinositides and NF2 in Hippo pathway regulation.

The Hippo pathway is a master regulator of tissue homeostasis and organ size. NF2 is a well-established tumor suppressor, and loss of NF2 severely compromises Hippo pathway activity. However, the precise mechanism of how NF2 mediates upstream signals to regulate the Hippo pathway is not clear. Here we report that, in mammalian cells, NF2's lipid-binding ability is critical for its function in activating the Hippo pathway in response to osmotic stress. Mechanistically, osmotic stress induces PI(4,5)P2 plasma membrane enrichment by activating the PIP5K family, allowing for NF2 plasma membrane recruitment and subsequent downstream Hippo pathway activation. An NF2 mutant deficient in lipid binding is unable to activate the Hippo pathway in response to osmotic stress, as measured by LATS and YAP phosphorylation. Our findings identify the PIP5K family as novel regulators upstream of Hippo signaling, and uncover the importance of phosphoinositide dynamics, specifically PI(4,5)P2, in Hippo pathway regulation.

RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation.

Both the timing and kinetics of neurotransmitter release depend on the positioning of clustered Ca2+ channels in active zones to docked synaptic vesicles on presynaptic plasma membranes. However, how active zones form is not known. Here, we show that RIM and RIM-BP, via specific multivalent bindings, form dynamic and condensed assemblies through liquid-liquid phase separation. Voltage-gated Ca2+ channels (VGCCs), via C-terminal-tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM and RIM-BP condensates. We further show that RIM and RIM-BP, together with VGCCs, form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of active zones, and the formation of RIM and RIM-BP condensates may cluster VGCCs into nano- or microdomains and position the clustered Ca2+ channels with Ca2+ sensors on docked vesicles for efficient and precise synaptic transmissions.

CaMKII autophosphorylation is the only enzymatic event required for synaptic memory.

Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of target protein phosphorylation. Thus, autophosphorylation and binding to the GluN2B subunit are the only two requirements for CaMKII in synaptic memory.

Field-dependent deep learning enables high-throughput whole-cell 3D super-resolution imaging.

Single-molecule localization microscopy in a typical wide-field setup has been widely used for investigating subcellular structures with super resolution; however, field-dependent aberrations restrict the field of view (FOV) to only tens of micrometers. Here, we present a deep-learning method for precise localization of spatially variant point emitters (FD-DeepLoc) over a large FOV covering the full chip of a modern sCMOS camera. Using a graphic processing unit-based vectorial point spread function (PSF) fitter, we can fast and accurately model the spatially variant PSF of a high numerical aperture objective in the entire FOV. Combined with deformable mirror-based optimal PSF engineering, we demonstrate high-accuracy three-dimensional single-molecule localization microscopy over a volume of ~180 × 180 × 5 µm3, allowing us to image mitochondria and nuclear pore complexes in entire cells in a single imaging cycle without hardware scanning; a 100-fold increase in throughput compared to the state of the art.

The INAD scaffold is a dynamic, redox-regulated modulator of signaling in the Drosophila eye.

INAD is a scaffolding protein that regulates signaling in Drosophila photoreceptors. One of its PDZ domains, PDZ5, cycles between reduced and oxidized forms in response to light, but it is unclear how light affects its redox potential. Through biochemical and structural studies, we show that the redox potential of PDZ5 is allosterically regulated by its interaction with another INAD domain, PDZ4. Whereas isolated PDZ5 is stable in the oxidized state, formation of a PDZ45 "supramodule" locks PDZ5 in the reduced state by raising the redox potential of its Cys606/Cys645 disulfide bond by ∼330 mV. Acidification, potentially mediated via light and PLCß-mediated hydrolysis of PIP(2), disrupts the interaction between PDZ4 and PDZ5, leading to PDZ5 oxidation and dissociation from the TRP Ca(2+) channel, a key component of fly visual signaling. These results show that scaffolding proteins can actively modulate the intrinsic redox potentials of their disulfide bonds to exert regulatory roles in signaling.

New targets and designed inhibitors of ASAP Arf-GAPs derived from structural characterization of the ASAP1/440-kD ankyrin-B interaction.

ASAP1 and its paralog ASAP2 belong to a PI4,5P2-dependent Arf GTPase-activating protein (Arf-GAP) family capable of modulating membrane and cytoskeletal dynamics. ASAPs regulate cell adhesive structures such as invadosomes and focal adhesions during cell attachment and migration. Malfunctioning of ASAP1 has been implicated in the malignant phenotypes of various cancers. Here, we discovered that the SH3 domain of ASAP1 or ASAP2 specifically binds to a 12-residue, positively charged peptide fragment from the 440 kDa giant ankyrin-B, a neuronal axon specific scaffold protein. The high-resolution structure of the ASAP1-SH3 domain in complex with the gAnkB peptide revealed a noncanonical SH3-ligand binding mode with high affinity and specificity. Structural analysis of the complex readily uncovered a consensus ASAP1-SH3 binding motif, which allowed the discovery of a number of previously unknown binding partners of ASAP1-SH3 including Clasp1/Clasp2, ALS2, ß-Pix, DAPK3, PHIP, and Limk1. Fittingly, these newly identified ASAP1 binding partners are primarily key modulators of the cytoskeletons. Finally, we designed a cell-penetrating, highly potent ASAP1 SH3 domain binding peptide with a Kd ∼7 nM as a tool for studying the roles of ASAPs in different cellular processes.

Abnormal Thalamo-Cortical Interactions in Overlapping Communities of Migraine: An Edge Functional Connectivity Study.

OBJECTIVE: Migraine has been demonstrated to exhibit abnormal functional connectivity of large-scale brain networks, which is closely associated with its pathophysiology and has not yet been explored by edge functional connectivity. We used an edge-centric approach combined with motif analysis to evaluate higher-order communication patterns of brain networks in migraine. METHODS: We investigated edge-centric metrics in 108 interictal migraine patients and 71 healthy controls. We parcellated the brain into networks using independent component analysis. We applied edge graph construction, k-means clustering, community overlap detection, graph-theory-based evaluations, and clinical correlation analysis. We conducted motif analysis to explore the interactions among regions, and a classification model to test the specificity of edge-centric results. RESULTS: The normalized entropy of lateral thalamus was significantly increased in migraine, which was positively correlated with the baseline headache duration, and negatively correlated with headache duration reduction following preventive medications at 3-month follow-up. Network-wise entropy of the sensorimotor network was significantly elevated in migraine. The community similarity between lateral thalamus and postcentral gyrus was enhanced in migraine. Migraine patients showed overrepresented L-shape and diverse motifs, and underrepresented forked motifs with lateral thalamus serving as the reference node. Furthermore, migraine patients presented with overrepresented L-shape triads, where the postcentral gyrus shared different edges with the lateral thalamus. The classification model showed that entropy of the lateral thalamus had the highest discriminative power, with an area under the curve of 0.86. INTERPRETATION: Our findings indicated an abnormal higher-order thalamo-cortical communication pattern in migraine patients. The thalamo-cortical-somatosensory disturbance of concerted working may potentially lead to aberrant information flow and deficit pain processing of migraine. ANN NEUROL 2023;94:1168-1181.

Three-Dimensional Reconfigurable Optical Singularities in Bilayer Photonic Crystals.

Metasurfaces and photonic crystals have revolutionized classical and quantum manipulation of light and opened the door to studying various optical singularities related to phases and polarization states. However, traditional nanophotonic devices lack reconfigurability, hindering the dynamic switching and optimization of optical singularities. This paper delves into the underexplored concept of tunable bilayer photonic crystals (BPhCs), which offer rich interlayer coupling effects. Utilizing silicon nitride-based BPhCs, we demonstrate tunable bidirectional and unidirectional polarization singularities, along with spatiotemporal phase singularities. Leveraging these tunable singularities, we achieve dynamic modulation of bound-state-in-continuum states, unidirectional guided resonances, and both longitudinal and transverse orbital angular momentum. Our work paves the way for multidimensional control over polarization and phase, inspiring new directions in ultrafast optics, optoelectronics, and quantum optics.

Carbon monoxide as a negative feedback mechanism on HIF-1α in the progression of metabolic-associated fatty liver disease.

Metabolic-associated fatty liver disease (MAFLD) encompasses various chronic liver conditions, yet lacks approved drugs. Hypoxia-inducible factor-1α (HIF-1α) is pivotal in MAFLD development. Our prior research highlighted the efficacy of the nano-designed carbon monoxide (CO) donor, targeting HIF-1α in a mouse hepatic steatosis model. Given heme oxygenase-1 (HO-1, a major downstream molecule of HIF-1α) as the primary source of intrinsic CO, we hypothesized that upregulation of HO-1/CO, responsive to HIF-1α, forms a negative feedback loop regulating MAFLD progression. In this study, we explored the potential negative feedback mechanism of CO on HIF-1α and its downstream effects on MAFLD advancement. HIF-1α emerges early in hepatic steatosis induced by a high-fat (HF) diet, triggering increased HO-1 and inflammation. SMA/CORM2 effectively suppresses HIF-1α and steatosis progression when administered within the initial week of HF diet initiation but loses impact later. In adipose tissues, concurrent metabolic dysfunction and inflammation with HIF-1α activation suggest adipose tissue expansion initiates HF-induced steatosis, triggering hypoxia and liver inflammation. Notably, in an in vitro study using mouse hepatocytes treated with fatty acids, downregulating HO-1 intensified HIF-1α induction at moderate fatty acid concentrations. However, this effect diminished at high concentrations. These results suggest the HIF-1α-HO-1-CO axis as a feedback loop under physiological and mild pathological conditions. Excessive HIF-1α upregulation in pathological conditions overwhelms the CO feedback loop. Additional CO application effectively suppresses HIF-1α and disease progression, indicating potential application for MAFLD control.

Profile of Chinese Cluster Headache Register Individual Study (CHRIS): Clinical characteristics, diagnosis and treatment status data of 816 patients in China.

BACKGROUND: The clinical profile of cluster headache may differ among different regions of the world, warranting interest in the data obtained from the initial Chinese Cluster Headache Register Individual Study (CHRIS) for better understanding. METHODS: We conducted a multicenter, prospective, longitudinal cohort study on cluster headache across all 31 provinces of China, aiming to gather clinical characteristics, treatment approaches, imaging, electrophysiological and biological samples. RESULTS: In total 816 patients were enrolled with a male-to-female ratio of 4.33:1. The mean age at consultation was 34.98 ± 9.91 years, and 24.89 ± 9.77 years at onset. Only 2.33% were diagnosed with chronic cluster headache, and 6.99% had a family history of the condition. The most common bout was one to two times per year (45.96%), lasting two weeks to one month (44.00%), and occurring frequently in spring (76.23%) and winter (73.04%). Of these, 68.50% experienced one to two attacks per day, with the majority lasting one to two hours (45.59%). The most common time for attacks was between 9 am and 12 pm (75.86%), followed by 1 am and 3 am (43.48%). Lacrimation (78.80%) was the most predominant autonomic symptom reported. Furthermore, 39.22% of patients experienced a delay of 10 years or more in receiving a correct diagnosis. Only 35.67% and 24.26% of patients received common acute and preventive treatments, respectively. CONCLUSION: Due to differences in ethnicity, genetics and lifestyle conditions, CHRIS has provided valuable baseline data from China. By establishing a dynamic cohort with comprehensive multidimensional data, it aims to advance the management system for cluster headache in China.

Myosin VI undergoes cargo-mediated dimerization.

Myosin VI is the only known molecular motor that moves toward the minus ends of actin filaments; thus, it plays unique roles in diverse cellular processes. The processive walking of myosin VI on actin filaments requires dimerization of the motor, but the protein can also function as a nonprocessive monomer. The molecular mechanism governing the monomer-dimer conversion is not clear. We report the high-resolution NMR structure of the cargo-free myosin VI cargo-binding domain (CBD) and show that it is a stable monomer in solution. The myosin VI CBD binds to a fragment of the clathrin-coated vesicle adaptor Dab2 with a high affinity, and the X-ray structure of the myosin VI CBD in complex with Dab2 reveals that the motor undergoes a cargo-binding-mediated dimerization. The cargo-binding-induced dimerization may represent a general paradigm for the regulation of processivity for myosin VI as well as other myosins, including myosin VII and myosin X.

Evaluation of the quality of products from multiple industrial-scale composting treatment facilities for kitchen waste and exploration of influencing factors.

The aerobic composting process is extensively utilized to manage kitchen waste. Nonetheless, the variability in the quality of compost derived from engineering practices which significantly hinders its broader industrial application. This work investigated the final products of kitchen waste compost at multiple industrial-scale treatment facilities utilizing three distinct aerobic composting processes in a bid to explore key factors affecting compost quality. The quality evaluation was based on technical parameters like seed germination index (GI), and limiting factors such as heavy metal content. The results showed that most of the compost products failed to meet the established standards, with GI being the primary limiting indicator. Furthermore, maturity assessments suggested that compost with low GI exhibited reduced humification could not be recommended for agricultural use. The investigation delved into the primary determinants of GI, focusing on risk factors such as the oil and salt of kitchen waste, and the microbial community of the humification driving forces. The results indicated that products with low GI had higher oil and salt content and a relatively simple microbial community. A thorough analysis suggested that excessive levels oil and salt were potential influencing factors on GI, as they stimulated the activity of acid-producing bacteria like Lactobacillus, suppressed the activity of humification-promoting bacteria such as Actinomarinales, and influenced the decomposition and humification processes of organic matter and total nitrogen, thereby affecting product quality. The findings provide valuable insights for improving kitchen waste compost products for agricultural applications.